Methylene blue for the treatment or prophylaxis of encephalopathy caused by ifosfamide

ABSTRACT

The encephalopathy caused by ifosfamide and similarly acting compounds can be prevented or treated by the administration of methylene blue or another compound which is able to oxidize a reduced flavin moiety. Pyritinol is able to reduce other aspects of ifosfamide toxicity.

FIELD OF THE INVENTION

This invention relates to pharmaceutical uses of compounds and topharmaceutical compositions. In particular, the invention relates to thetreatment or prophylaxis of encephalopathy, as may be caused by certainphosphoramide compounds.

BACKGROUND OF THE INVENTION

Ifosfamide(N,3-bis(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine2-oxide) ##STR1## is a phosphoramide compound which is a well knownantineoplastic drug. It is a structural isomer of cyclophosphamide(N,N-bis(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine2-oxide) ##STR2## which is also a phosphoramide compound and has asimilar clinical utility. Although the antineoplastic activities ofifosfamide and cyclophosphamide are similar, there are sufficientdifferences for ifosfamide to be useful in situations which have provedrefractory to cyclophosphamide therapy.

Ifosfamide is strictly speaking a pro-drug: it has to be metabolised inthe liver to be active. In view of this, orally administered ifosfamidewould be expected to be more active than the parenterally administeredcompound; and indeed it is. Oral administration of ifosfamide isdesirable not only for the clinical reason of improved bio-activity ofthe drug but also for the economic reason that non-parenteraladministration would reduce hospitalisation. Unfortunately, at leastsome of ifosfamide's toxic effects are also more pronounced on oraladministration, again because of the effects of its metabolites.Surprisingly, until now the biochemical activities by whichcyclophosphamide and in particular ifosfamide form their extremely toxicmetabolites has remained illusive. It should be appreciated that thegroup of pharmaceuticals known as oxazaphospharines, such as ifosfamide,cyclophosphoramide and bromoifosfamide, are optically active compoundsand therefore any reference made throughout this disclosure, forexample, to ifosfamide, is also intended to include the R and S isomersof ifosfamide unless the context requires otherwise.

As with many effective antineoplatic drugs, the toxicity of ifosfamideis clinically a very real problem. Ifosfamide causes haemorrhagiccystitis, a severe toxicity to the bladder, (as does, to a lesserextent, cyclophosphamide) and is practically always administered inconjunction with mesna (sodium 2-mercaptoethanesulphonate), which has auroprotective action against the effects of both ifosfamide andcyclophosphamide. Although the urinary tract toxicity can therefore nowadequately be managed, the same cannot be said for other toxic effectsof ifosfamide, particularly its effects on the central nervous system(CNS).

Martindale (28th Edition, 1982) reports that about 10% of patientsreceiving intravenous ifosfamide experience CNS side effects, especiallyconfusion and lethargy. When ifosfamide is administered orally, theposition is even worse: despite its improved efficacy against manytumour types when administered orally, the drug cannot be ethicallyjustified for oral administration, in view of the CNS side effects. Infact, it has been reported that Asta Medica of Frankfurt, who developedifosfamide, have ceased to sponsor clinical trials of oral formulationsof the drug, largely because of the unwillingness of regulatoryauthorities to grant clinical trial certificates. Even when administeredparenterally, the use of ifosfamide at high doses and in patients withorgan dysfunction (such as renal failure) is contraindicated because ofthe frequent occurrence of CNS toxicity.

The CNS toxicity of ifosfamide manifests itself as encephalopathy; inother words, patients exhibit signs of cerebral irritation without anylocalised lesion to account for them. The cerebral irritation isapparent as cognitive, as opposed to motor or sensory dysfunction, andthe symptoms experienced by patients include, in addition to theconfusion and lethargy referred to above, sleep disturbance,hallucinations, psychoses and often frank coma.

To date, the only option to the clinical oncologist, if ifosfamideencephalopathy should occur has been the withdrawal of ifosfamidetherapy, with the inevitable consequence for the progression ormetastasis of the tumour. So there has existed for some time a need foreffective means for monitoring and, especially, preventing and treatingifosfamide encephalopathy. In spite of past efforts, the mechanism ofifosfamide encephalopathy, the factors affecting it and the means forcontrolling it have proved illusive. Vast numbers of cancer patients whocould otherwise benefit from ifosfamide chemotherapy are presentlyexcluded from treatment.

SUMMARY OF THE INVENTION

The present invention relates to a solution to this problem. It has beendiscovered that toxic effects of encephalopathy-causing compounds suchas ifosfamide are due to interference with one or more flavoproteins orother components of the respiratory chain. Toxicity can therefore beavoided or ameliorated by administering compounds which reduce theeffect of that interference. In particular, it has been discovered thatmethylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride) andcompounds which act similarly to methylene blue, including methyleneblue in its reduced form: leucomethylene blue, can prevent or treattoxicity, especially encephalopathy, caused by such compounds asifosfamide.

DETAILED DESCRIPTION

According to a first aspect of the invention, there is provided the useof a first compound in the preparation of a medicament for preventing ortreating toxicity caused by a second compound, wherein the firstcompound is able to oxidise a reduced flavin moiety and wherein thesecond compound is ifosfamide or another compound which causes toxicityin a manner similar to ifosfamide. The invention therefore hasapplication in a method of treating or preventing toxicity caused byifosfamide or another compound which causes toxicity in a manner similarto ifosfamide, the method comprising administering to a subject aneffective amount of a compound which is able to oxidise a reduced flavinmoiety.

The compounds whose toxicity the present invention is useful inalleviating include ifosfamide and all those compounds which causetoxicity, particularly encephalopathy, in a manner similar toifosfamide. Many such compounds are also antineoplastic or cytotoxicagents useful in cancer chemotherapy. More specifically, the inventionis useful in alleviating encephalopathy caused by phosphoramidecompounds. Certain of those phosphoramide compounds, includingifosfamide itself, are disclosed in GB-A-1188159 and fall within generalformula I: ##STR3## wherein R₁ represents C₁ -C₄ alkyl substituted byone or more halogen atoms, X₁ represents an oxygen or sulphur atom, Yrepresents an oxygen atom or a group of the formula --NH-- or --NZ₁ --in which Z₁ represents C₁ -C₄ alkyl optionally substituted by one ormore halogen atoms or hydroxy groups, each Z independently representshydrogen, halogen, C₁ -C₄ alkyl, hydroxy or hydroxymethyl, m represents2 or 3 and X₂ represents an ethyleneimine group or a group of thegeneral formula Ia: ##STR4## wherein R represents hydrogen or C₁ -C₄alkyl optionally substituted by one or more halogen atoms or hydroxygroups, X represents a halogen atom or hydroxy group and Z and m havethe meanings given above.

Preferred alkyl groups are ethyl, and preferred halogen atoms arebromine and, especially, chlorine. When Z is not hydrogen it ispreferably 2-chloroethyl or 3-chloropropyl. X₁ is preferably oxygen.Other preferences include those referred to in GB-A-1188159: forexample, preference is given to compounds of general formula II:##STR5## wherein R₁, X₂ and m have the same meaning as for generalformula I. Most preferred are compounds of general formula III: ##STR6##wherein R₂ represents a 2-chloroethyl or 3-chloropropyl group(preferably 2-chloroethyl), and R₃ represents hydrogen or a methyl orethyl group, possibly substituted in the 2-position with hydroxy or,preferably, chlorine.

The invention is expected to be particularly useful in conjunction withifosfamide therapy.

Other phosphoramide compounds which cause encephalopathy include TEPA(1,1',1"-phosphinylidynetrisaziridine) and thio-TEPA(1,1',1"-phosphinothioylidynetrisaziridine), as shown in general formulaIV: ##STR7## wherein Q represents oxygen for TEPA and sulphur forthio-TEPA.

Analogues of thio-TEPA having included compounds of general formula V:##STR8## in which each R independently is hydrogen or C₁ -C₄ alkyl andeach of R' and R" independently is hydrogen or C₁ -C₄ alkyl or, whentaken together with the nitrogen atom to which they are attached, R' andR" form a saturated heterocyclic radical containing from 3 to 6 atoms.

It is believed that the encephalopathic toxicity of ifosfamide andsimilarly toxic compounds is due to the formation of certain haloaceticacids, particularly chloracetic acid, and certain aldehyde metabolites.For example, the 2-chloroethyl group at the 3-position of the ifosfamidering is believed to be metabolised, probably by cytochrome P450 in theliver or elsewhere, to chloracetaldehyde (ClCH₂ CHO), which issubsequently oxidised to chloracetic acid (ClCH₂ COOH).Chloracetaldehyde is putatively a strong encephalopathic metabolite ofifosfamide. Chloracetic acid is also a potent acetylating and alkylatingagent, with an affinity for sulphur in oxidation state II. It may alsobe a substrate for enzymes in the tricarboxylic acid cycle, generatinghighly toxic chlorocitric acid and other halogenated metabolites.

Other compounds which similarly are reactive with sulphur II includeaziridine-containing compounds, that is to say compounds which containthe structure: ##STR9## As well as going some way to explain thetoxicity of thio-TEPA and related compounds, this observation may alsoplay a part in explaining ifosfamide toxicity, as a 2-chloroethylaminomoiety (particularly a primary 2-chloroethylamino moiety, as present inthe 3-position in ifosfamide but absent in cyclophosphamide) may reactto form an aziridine ring.

It is therefore possible that ifosfamide encephalopathy and similarencephalopathies can now be explained on the basis of reaction withsulphur II, or other sites, in critical enzymes or enzyme cofactors. Forexample, in the electron transport chain which gives rise to oxidativephosphorylation at the mitochondrial membrane, iron-sulphur centres("non-haem iron") play a crucial role. These complexes are held inplace, for instance in the flavin-containing NADH dehydrogenase complex("Complex I"), by cysteine residues. If ifosfamide metabolites disruptiron-sulphur centres, whether by reacting with the anchoring cysteineresidues or by reacting with the sulphur in the iron-sulphur centreitself, so as to prevent or reduce the electron transport role offlavin, then the electron transport chain would become blocked and ATPgeneration would be severely attenuated. While the consequences ofgreatly reduced ATP generation are serious for the body as a whole, theconsequences for the brain may be so severe as to account for theclinically observed encephalopathy.

The affinity of ifosfamide's metabolites for sulphur (II) may alsodeplete the cell's pool of coenzyme A (CoA), which is a thiol in itsnon-esterified form. As CoA is essential for supplying carbon atoms tothe tricarboxylic acid cycle, and from the β-oxidation of fatty acids,both of which feed into the respiratory electron transport chain, theoverall effect may be synergistic.

The invention makes use of compounds which are able to oxidise a reducedflavin moiety, for example in a flavin-containing electron transferprotein or a flavin-linked oxidation-reduction enzyme. In particular,the enzyme will usually be a flavin dehydrogenase. One of the mostcrucial flavin dehydrogenases is NADH dehydrogenase or, more formally,NADH-ubiquinone oxidoreductase (Complex I) in the electron transportchain. This enzyme complex accepts electrons from NADH (generated forexample by glycolysis and the tricarboxylic acid cycle), therebyoxidising NADH to NAD and reducing the flavin moiety. In turn, theelectrons are passed on, by means of an iron-sulphur centre, to coenzymeQ (ubiquinone) and the reduced flavin is reoxidised. Subsequently, theelectrons are transported, via

a second iron-sulphur centre,

a complex (Complex III) comprising cytochrome b (b₅₆₆ and b₅₆₂), a thirdiron-sulphur centre and cytochrome c₁,

cytochrome c and

a complex (Complex IV) of cytochromes a and a₁ to molecular oxygen,which is reduced to water.

In this electron transport or respiratory chain, hydrogen ions (protons)are transported across the mitochondrial membrane at complex I, complexIII and complex IV. The transmembrane proton-motive force established bythe resulting hydrogen ion gradient drives the phosphorylation of ADP toATP by the membrane-spanning enzyme F₁ ATPase.

The generation of ATP, the "loose change" of energy in the cell, istherefore dependent in aerobic organisms on the functioning of theelectron transport chain. If that chain is blocked at a sufficientlyearly stage to prevent even the first of the three "proton pumps"operating, the cell may rapidly cease proper functioning.

Other flavoproteins involved in the electron transfer or respiratorychain include electron transfer flavoprotein (ETF) and electron transferflavoprotein-ubiquinone oxidoreductase (ETF-QO). These proteins mediateelectron transfer from mitochondrial oxidation of fatty acids andvarious amino acids as well as the catabolism of choline (viaN,N-dimethyl glycine and sarcosine) to the main respiratory chain. Theeffect of them is to produce reduced ubiquinone (QH₂) to join the QH₂produced by Complex I, as described above.

Genetic deficiencies in ETF and ETF-QO are known to be the primarycauses of glutaric acidaemia type II, an inborn error characterisedclinically by non-ketotic hypoglycaemia and metabolic acidosis. Glutaricacid and sarcosine are found in the urine of glutaric acidaemic type IIpatients. It was the observation of these same two substances in theurine of patients suffering ifosfamide encephalopathy which was one ofthe keys to the present invention; the invention may be regarded, inpart, as being founded on the discovery that ifosfamide encephalopathyand similar encephalopathies are drug-induced equivalents of the geneticdisease. It may be that one of the primary functions of the invention isto restore proper, or at least adequate, electron flow at the site ofETF/ETF-QO.

A further flavoprotein involved in the respiratory or electron transportchain is FAD-containing glycerol phosphate dehydrogenase, which oxidisesglycerol phosphate (from cytoplasmic glycolysis) to dihydroxyacetonephosphate while reducing coenzyme Q to join the QH₂ pool. Complex II(succinate-ubiquinone oxidoreductase), which dehydrogenates succinicacid to fumaric acid, while reducing coenzyme Q to QH₂, also containsflavin as FAD.

Many other important enzymes, in both the mitochondrion and thecytoplasm, are flavoproteins, and their proper functioning may also bedisturbed by ifosfamide (or at least its metabolites). It may not matterwhether the flavoprotein is covalently or non-covalently bound to itsapoprotein; flavin is known to be bound in a variety of different waysand held in different electrostatic microenvironments, so that it mayhave a variety of different redox potentials in different circumstances.

Chlorethylamine, another metabolite of ifosfamide, would be a substratefor monoamine oxidase, another mitochondrial flavoprotein, by which itwould be oxidised to chloracetaldehyde and chloracetic acid, whose toxiceffects have already been discussed in detail.

As was mentioned above, ifosfamide is also known to be metabolised intovarious aldehydes in the liver, namely chloracetaldehyde, acrolein (CH₂═CH--CHO), and aldoifosfamide. Acrolein is also a well known metaboliteof cyclophosphamide and is extremely toxic to the bladder causinghaemorrhagic cystitis. It is postulated that when ifosfamide isadministered orally, these aldehyde metabolites super-saturate theavailable supply of hepatic aldehyde dehydrogenases, thereby depletingNAD stores with a coinciding build-up in NADH levels. Accordingly,without available dehydrogenases, these toxic aldehyde metabolitesdepart the liver exerting various systemic toxicities, includingencephalopathy, in the human body. It may well be that an importantfunction of this invention is simply to prevent the accumulation oftoxic aldehyde metabolites, such as chloracetaldehyde, in the liver andthereby preventing exertion of their toxic effects, includingencephalopathy, systemically throughout the body.

In sum, it can be seen that there are many vulnerable flavin-containingenzymes and other flavoproteins in the cell, particularly in themitochondrial membrane, whose proper functioning is vital to the cell.Their prolonged disruption could be fatal.

Methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride) hasthe structure ##STR10## and is able to oxidise a reduced flavin moiety,thus freeing it from being locked in its reduced form. It has been usedin an attempt to treat congenital glutaric acidaemic type II (Frerman &Goodman, "Glutaric Acidemia Type II and Defects of the MitochondrialRespiratory Chain", Chapter 34 in "The Metabolic Basis of InheritedDisease", 6th Edition, McGraw-Hill, 1989 and Harpey et al. The Lancet,1:391 (1986)). Methylene blue is also administered as a means forpreventing ethanol-induced hypoglycemia by oxidising NADH to NAD⁺ andrestoring hepatic glucose output depressed by high ethanol consumption.Madison et al., Diabetes, 16:252-258 (April, 1967). It is believed thatmethylene blue is acting in a similar fashion in its prevention andtreatment for the onset of ifosfamide encephalopathy.

As discussed previously, after the oral administration of ifosfamide,chloracetaldehyde or other toxic ifosfamide metabolites are formed inthe liver. In order for the liver to process these metabolites, variousflavin-dependent dehydrogenase enzymes are required. Additionally, sinceit is believed that ifosfamide saturates the aldehyde dehydrogenaseenzymes in the liver thereby lowering the supply of NAD available,encephalopathy results. Methylene blue however, is able to treat as wellas prevent this toxicity due to its redox potential. The methylene bluecauses NADH to be oxidised to NAD⁺ thereby simultaneously activating thealdehyde dehydrogenases in the liver. These activated aldehydedehydrogenase enzymes ultimately break down the ifosfamide metabolitesand decrease significantly the levels of circulating ifosfamidemetabolites which can cause systemic toxicity.

It is also a proposed that methylene blue is acting as a type ofchlorinating agent, chlorinating the toxic ifosfamide metabolites andthereby forming non-toxic metabolites that are excretable in urine. Thisproposal comes from the identification of three surprising and novelurinary metabolites found among those patients treated with bothifosfamide and methylene blue. These metabolites are2,2,2-trichlorethanol (CCl₃ CH₂ OH), trichloracetic acid (CCl₃ COOH),and 3-chlorproprionic acid (ClCH₂ CH₂ COOH). The first two metabolitesarise by the dichlorination of chloracetaldehyde. Trichloroethanol inrather large doses is a known sedative compound. The last metabolite,3-chloroproprionic acid, is believed to be formed by the addition of achlorine atom to acrolein. It is postulated that when methylene blue isreduced to leucomethylene blue in the human body, that hydrogen peroxideis generated. The hydrogen peroxide is readily able to interact withchloride ions (Cl⁻) forming hypochloric acid (HOCl). Hypochloric acid isalso a powerful chlorinating agent. It is believed that the hypochloricacid formed by the metabolism of methylene blue, sequentially adds twochlorine atoms to the β-carbon of chloracetaldehyde, chlorethanol andchloracetic acid to ultimately yield trichlorinated, non-toxicifosfamide derivatives. These ifosfamide derivatives are subsequentlyexcreted in the urine. It is also thought the hypochloric acid formed bythe metabolism of methylene blue also adds one chlorine atom at thedouble-bond of acrolein yielding 3-chloroproprionic acid which is alsonon-toxic to the human body and is readily excreted in urine. Analysisof patient urine substantiates these findings.

It is further believed that methylene blue is preventing the onset ofencephalopathy by inhibiting the functioning of plasma amine oxidases.The plasma amine oxidases readily convert 2-chloroethylamine tochloracetaldehyde. Neumann et al., The Journal of Biological Chemistry,260: 6362-6367 (1975). Methylene blue is therefore believed to preventthe accumulation of the various ifosfamide metabolites in the liverby: 1) oxidising reduced flavin moieties hence activating aldehydedehydrogenase enzymes in the liver; 2) acting as a rapid chlorinatingagent; and 3) by inhibiting plasma amine oxidases and thus preventingthe conversion of 2-chlorethylamine to chloracetaldehyde. Analysis ofpatient urine also substantiates the proposal regarding inhibition ofplasma amine oxidases.

Other compounds which may be useful in the practice of the inventioninclude riboflavin, other flavins and flavoproteins, phenazinemethosulphate, ferricyanides such as sodium ferricyanide, andubiquinone, as they are able to oxidise reduced flavin in variouscircumstances. Compounds useful in the invention will generally besupplied in oxidised form, but it may be feasible for them to beadministered in reduced form for oxidation by the body's endogenousoxidising agents.

A particularly preferred embodiment of the invention is the use ofmethylene blue in the preparation of a medicament for preventing ortreating ifosfamide encephalopathy. The invention therefore hasapplication in a method of treating or preventing ifosfamideencephalopathy, the method comprising administering to a subject aneffective amount of methylene blue. Structural analogues and derivativesof methylene blue in which the redox potential and/or other chemicalproperties are not significantly disturbed are also useful in thisinvention and are included within the term "methylene blue" unless thecontext requires otherwise.

According to a second aspect of the invention, there is provided aproduct comprising a first compound and a second compound, wherein thefirst compound is able to oxidise a reduced flavin moiety and whereinthe second compound is ifosfamide or another compound which causestoxicity in a manner similar to ifosfamide, as a combined preparationfor simultaneous, separate or sequential use in cancer chemotherapy orother therapy involving the second compound. A preferred embodiment ofthis aspect of the invention provides a product comprising ifosfamideand methylene blue as a combined preparation for simultaneous, separateor sequential use in cancer chemotherapy.

According to a third aspect of the invention, there is provided apharmaceutical formulation comprising a first compound and a secondcompound, wherein the first compound is able to oxidise a reduced flavinmoiety and wherein the second compound is ifosfamide or another compoundwhich causes toxicity in a manner similar to ifosfamide, and optionallya pharmaceutically acceptable carrier. A preferred embodiment of thisaspect of the invention provides a pharmaceutical formulation comprisingifosfamide and methylene blue, and optionally a pharmaceuticallyacceptable carrier.

In the practice of the preferred embodiment of the invention, in itsvarious aspects, ifosfamide may be administered parenterally (forexample by intravenous injection or, more usually, infusion) but ispreferably administered orally. Ifosfamide dosages are conventionallymeasured in terms of grams of drug per square meter of the patient'stotal body surface area. (The body surface area of a 70 kg adult male istypically about 1.7 m².) In a four to six day cycle of intravenouslyadministered ifosfamide, a total dosage for treatment of 10 to 18 g/m²may typically be given. The ifosfamide is administered once a day forthe duration of the cycle. Orally, ifosfamide has been administered atabout 3 to 5 g/m² over a treatment cycle. These dosages are not expectedto be limiting on the present invention; indeed, the invention may makeit possible for the patient to receive higher doses than has hithertobeen considered reasonable. Also, although continuous infusion ofifosfamide has been found to give rise to fewer instances ofencephalopathy than injection, it may be that, by means of the presentinvention, continuous infusion may be dispensed with in a greater numberof cases; even if the patient still has to be hospitalised, he maytherefore at least be ambulatory. In all cases, the actual doseadministered and the method of administration will be at the discretionof the clinical oncologist or other physician.

Ifosfamide therapy is in practice not contemplated without treatment toreduce the severe bladder toxicity of the drug. Mesna (sodium2-mercaptoethanesulphonate) is usually used for this purpose. Typically,mesna is administered at 60-100% (w/w) of the total dose of ifosfamide.Mesna treatment is usually started with or just before the first dose ofifosfamide, at which time a 20% dose may be given; subsequent doses of20% may then be given every few hours. Mesna is usually administered byintravenous injection. Oral administration might be preferred, giventhat the patient may be receiving a variety of other medicaments byinjection or infusion, but mesna has a foul taste. If it is givenorally, attempts are usually made to disguise the taste, such as byformulating it in a soft drink, such as cola, or something stronger,such as whisky. On oral administration, the mesna dose is usuallydoubled, to compensate for bioavailability losses.

Other compounds which reduce toxic symptoms of ifosfamide may beadministered instead of or as well as mesna. For example, pyritinol, hasthe following chemical formula: ##STR11## and is the dimer of the thiolanalogue of pyridoxine (a derivative of vitamin B₆), and may be used asan ifosfamide detoxificant as it is believed to have a similar action tomesna but is sufficiently lipophilic to cross the blood-brain barrier.The use of pyritinol in reducing ifosfamide (and other phosphoramide,including cyclophosphamide) toxicity forms an independent part of theinvention.

According to a fourth aspect of the invention, there is provided the useof pyritinol in the preparation of a medicament for reducing orpreventing phosphoramide, particularly ifosfamide or cyclophosphamide,toxicity.

According to a fifth aspect of the invention, there is provided aproduct comprising pyritinol and a phosphoramide, particularlyifosfamide or cyclophosphamide, as a combined preparation forsimultaneous, separate or sequential use in chemotherapy, particularlycancer chemotherapy.

According to a sixth aspect of the invention, there is provided apharmaceutical formulation comprising pyritinol and a phosphoramide,particularly ifosfamide or cyclophosphamide, and optionally apharmaceutically acceptable carrier.

It is believed that pyritinol elevates intracellular thiolconcentrations thereby detoxifying the chloroethyl metabolites formed byifosfamide. Pyritinol is metabolised by reduction of its centraliseddisulfide bond, presumably by glutathione reductase or a similar enzymeusing GSH as its cofactor. Pyritinol is also known to be metabolised byS-methylation and S-oxidation into the following metabolites: ##STR12##These metabolites are the excretable forms of pyritinol in urine. Inpatients not receiving ifosfamide treatment, virtually 100% of pyritinolwill be excreted in its metabolised form into the urine. Uponadministration of ifosfamide however pyritinol is excreted solely in itsunmetabolised non-reduced form. This surprising result further supportsthe belief that ifosfamide treatment depletes the intracellular storesof free thiols by significantly lowering levels of available NADH andGSH in the respiratory chain. As was previously mentioned, without GSHas a cofactor, glutathione reductase is unable to reduce pyritinol intoits metabolised form. It is therefore a further aspect of this inventionto use the excretion of unmetabolised pyritinol as a biochemical markerfor the onset of encephalopathy upon administration of ifosfamide. Ithas also been demonstrated that metabolised forms of pyritinol willsubsequently be excreted in the urine upon addition of methylene blue,leucomethylene blue or similar compound to the schedule of treatment,further supporting the belief that methylene blue restores mitochondrialfunctions to normality.

Cytotoxic drugs such as ifosfamide are often given in combination withothers. Typical cytotoxic drugs administered in combination withifosfamide include adriamycin and cisplatin, particularly, but othersmay be used. Examples include cytosine arabinoside (araC), dacarbazine,epirubicin, VP16, vinca alkaloids such as vincristine and vinblastine,bleomycin, mitomycin C, BCNU, CCNU, 5-fluorouracil and methotrexate.Usual doses and modes of administration of these other cytotoxic drugswould often be used in the practice of the present invention, but againthe clinician's judgement would be the final determinant.

Ifosfamide, like many other cytotoxic drugs, causes severe emesis.Consequently, ifosfamide and ifosfamide-containing cocktails, areusually administered in conjunction with an antiemetic. There arevarious known classes of antiemetics, and the invention is notrestricted to the use of any of them. Example s of suitable antiemeticsinclude serotonin (5-HT₃) antagonists and dopamine D₂ antagonists suchas metaclopramide. Newer antiemetics include ondansetron, tropizatronand novoban. Parenterally, tropizatron is usually administered as ashort intravenous infusion, whereas the others are often given as slowintravenous injections. Orally, antiemetics are generally administeredin tablet form.

Ifosfamide is conventionally administered either in saline, fructose orglucose solution. In the present invention, the use of glucose is apreferred solution. The use of fructose is the most preferred solutionof the present invention, however, any solution containing a five or sixcarbon sugar moiety that would act as an energy source such as acetylglycerol may be used. It is well known that patients with glutaricaciduria type II require the administration of glucose to compensate forderangements in fatty-acid oxidation. The accompanying deficiency ofglucoeneogenisis in glutaric aciduria is specifically caused by theabsence of electron transferring flavoproteins. As with those patientssuffering from glutaric aciduria, it is believed that theco-administration of glucose, and in particular fructose, in ifosfamidepatients reduces the body's need for gluconeogenesis. The administrationof fructose results in augmented hepatic gluconeogenesis and furtherenhances the oxidation of NADH to NAD. The reoxidation of NAD isbelieved a vital step in overcoming the effects of encephalopathy.Additionally or alternatively, acetate generated by the action ofglycolysis on glucose may serve to dilute out the chloroacetic acidmetabolites of ifosfamide. Fructose may be administered orally at amaximum daily dosage of 300 grams and glucose may be administered orallyas a 5% solution with a maximum daily dosage of 100 grams.

It is often the case that patients who are undergoing ifosfamide therapywill be in severe pain because of the tumour or tumours being treated.They may therefore be taking painkillers such as morphine (for exampleas morphine sulphate tablets) or diacetyl morphine.

In the preferred embodiments of the invention, patients receivemethylene blue to counteract encephalopathy induced by ifosfamide.Methylene blue may be administered prophylactically, with the aim ofpreventing encephalopathy, or therapeutically, to rescue a patient froman encephalopathic episode. The literature dosages of methylene blue,when it is being used as a detoxificant, may be used as a guide tosuitable doses for use in the invention: from 2 to 3 mg/kg per 24 hourperiod may be suitable.

In accordance with the invention, methylene blue may be administeredorally or parenterally (for example by intravenous injection). The modeof administration will depend in part on whether methylene blue is beingadministered prophylactically or therapeutically; normally, oral dosagemay be preferred for prophylatic use, but the more rapid onset of actionprovided by parenteral, particularly intravenous, administration islikely to be more suitable for therapeutic rescue use, not least becausethe patient's ability to swallow may be impaired during encephalopathy.

For oral dosage, it is considered that from 200 to 400 mg per day isconsidered safe with 100 mg per day dosage increments to a maximumdosage of 500 mg per day. Caution must be used when administeringmethylene blue since excessive doses produce high quantities of hydrogenperoxide and may subsequently be chlorinated into hypocloric acid duringreduction to its leuco form. As was discussed previously, high levels ofhypochloric acid can ultimately produce trichloroethanol, which cancause a sedative effect in high dosages. In practice three or four 50 mgoral doses will usually be given. Methylene blue will usually be orallyadministered as a pharmacopoeia grade solid (for example to Ph. Eur. andPh. Helv. VII); hard gelatin capsules provide a suitable means of oraladministration.

For intravenous administration, the clinician may administer methyleneblue as a 2% w/v solution in water for injections or any other suitableexcipient. A 2 ml ampoule would contain 25 mg, so to provide a 50 mgdose three to four time a day the contents of an appropriate number ofpairs of ampoules would be used.

The invention has been described in general terms predominantly byreference to the presently perceived preferred embodiments, usingifosfamide and methylene blue or leucomethylene blue. Details of howother embodiments of the invention may be put into practice may bededuced by analogy. It is also to be understood that references madethroughout this specification to any theory explaining the resultsherein described is not to limit the scope of this invention. Preferredembodiments of each aspect of the invention are as for each otheraspect, mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be illustrated by the following non-limitingexamples wherein reference is made to the following figures:

FIG. 1 Gas Chromatography-Mass Spectrometry (GCMS) analysis of urinetaken from a patient undergoing chemotherapeutic treatment receivingifosfamide and methylene blue.

1A) 2,2,2-trichloroethanol (peak 1);

1B) 3-chloroproprionic acid (peak 2);

1C) Trichloroacetic acid (peak 3)

FIG. 2 Reference mass spectra of 2,2,2-Trichloroethanol generated fromFisons Autospec Q MASS LIB GCMS computer reference library at theDepartment of Organic Chemistry, University of Berne, Switzerland.

FIG. 3 Reference mass spectra of 3-chloroproprionic acid ethyl ester(3-chloroproprionic acid) generated from Fisons Autospec Q MASS LIB GCMScomputer reference library at the Department of Organic Chemistry,University of Berne, Switerland.

FIG. 4 Reference mass spectra of trichloracetic acid ethyl ester(trichloroacetic acid) generated from Fisons Autospec Q MASS LIB GCMScomputer reference library at the Department of Organic Chemistry,University of Berne, Switerland.

EXAMPLES Example 1

An 18 year old female patient diagnosed with advanced metastaticosteosarcoma began a five day chemotherapy regime. Treatment consistedof a total dosage of 12 g/m² ifosfamide with 80% (by weight, based onthe weight of ifosfamide) mesna and adriamycin (65 mg/m²) divided intotwo doses. She also received ZOFRAN™ brand ondansetron (antiemetic), 8mg, by intravenous injection and 200 mg pyritinol three times per dayorally. All parenteral medicaments were given in a total of 2 liters 5%glucose solution.

On day three of treatment, the patient had a bad night. She hadnightmares and displayed signs of encephalopathy-type hallucinations.The next morning she was still drowsy, disorientated and confused.

50 mg methylene blue, formulated at 2% w/v in water for injections, wasadministered by slow intravenous injection. After 1 hour, despitecontinued ifosfamide infusion, she was much calmer, coherent and able toarticulate the fearful nature of her hallucinations. The malaise andnausea which she had previously experienced were gone.

About 4 hours later, the 50 mg methylene blue dose was repeated toconsolidate the earlier treatment. That night she slept well and wasmuch better the next day. She was still tired, as is usual forifosfamide-treated patients, but showed no signs of confusion or othersymptoms of encephalopathy.

During this cycle of treatment, a nitrobenzyl pyridine (NBP) test of thepatient's urine for alkylating metabolites of ifosfamide showedpositive. This latter marker indicated that the ifosfamide was active inthe patient.

Example 2

After three weeks from the end of the first cycle, the patient who wasthe subject of Example 1 again was given the same chemotherapeuticregime, except that this time she received 50 mg oral capsules ofmethylene blue prophylactically three times a day for the full course oftreatment. She had no disturbed nights and responded well to thechemotherapy. A lung scan showed that the lung metastases were almostgone, showing the efficacy of the ifosfamide/adriamycin chemotherapy.

An NBP test on her urine showed that alkylating metabolites (presumablyof ifosfamide) were present.

Example 3

In 1990, an approximately 50 year old female patient with metastaticleiomyosarcoma was treated for lung metastases with ifosfamide,adriamycin and dacarbazine. She had three cycles of therapy at the time.In the first cycle, she became nervous and restless and exhibited mildencephalopatic signs (such as confusion and amnesia) short of psychosis.

In the second and third cycles, her dose of ifosfamide was 2 g/m² forthree days. In the second cycle, she became psychotic and somnolent. Inthe third cycle, she became severely psychotic and had hallucinationswhich continued not only after the end of the cycle but also afterdischarge from hospital. Apart from the side effects, the chemotherapywas successful, as it resulted in complete remission of the lungmetastases.

Between that ifosfamide treatment in 1990 and 1993, she underwentsurgery and radiotherapy at various times.

In August 1993, she was referred for ifosfamide treatment again. She hada tumour mass in her lower abdomen and metastases in the lung.

With the following treatment, she received four 50 mg doses of oralmethylene blue. Her cytotoxic chemotherapy comprised three 3.2 g dosesof ifosfamide (6 g/m² total dose) split over 3 days. The ifosfamide wasadministered as a continuous infusion with mesna. She also received 50mg/m² adriamycin in two separate doses on days 2 and 3. Additionally,she received pyritinol (200 mg) twice per day on each day of the fourday treatment.

The first cycle of treatment has been successfully completed. Despiteher history in 1990 of severe encephalopathy, no symptoms ofencephalopathy were recorded during treatment. She experienced nomalaise and only had one episode of vomiting.

Example 4

A male patient aged 51 years was diagnosed with non small cell lungcancer. He later developed brain, lung and mediastinal and bonemetastases. The course of treatment included surgery, irradiation of thebrain and chemotherapy. Dosage amounts for chemotherapy were as follows:ifosfamide (1 g/m²) given intravenously for days 1-3; mesna 60% ofintravenous ifosfamide dosage plus 800 mg orally twice daily for days1-3; VP-16 (100 mg) given orally for days 1-8 and methylene blue (50 mg)was administered orally as a loading dose on day 1, with 3×50 mgadministered orally on days 1-4. No signs of encephalopathy after fourcycles. The patient died 6 months later due to tumour progression.

Example 5

A 50 year old patient (female) was diagnosed with extensive small celllung cancer with metastases in the abdomen and exhibiting paraneoplasticlambert/Eaton Syndrome. She received six cycles of ifosfamide treatmentusing the identical dosage requirements as in Example 4. The patientexhibited no signs of encephalopathy and experienced good partialremission and the Lambert/Eaton Syndrome rapidly improved.

Example 6

A 62 year old female patient was diagnosed with non small cell lungcancer with extensive mediastinal lymphnode metastases. She wasoriginally prescribed a course of cisplatinum, VP-16 and velbe. However,no improvements in the disease appeared and the patient suffered fromsevere nausea, emesis and thrombocytopemia. Oral ifosfamide, VP-16, andmethylene blue was then ordered, using the same dosage requirements asdescribed in Example 4 above. Absolutely no signs of encephalopathy weredisplayed after six cycles of ifosfamide chemotherapy.

Example 7

A 64 year old patient (male) with non small cell lung cancer withinvolvment of the thoracic wall and retroperitoneal disease began onecycle of ifosfamide/methylene blue chemotherapy. No CNS toxicity wasexhibited using dosages as described in Example 4.

Example 8

A male patient, 66 years, was diagnosed with extensive small cell lungcancer with pleural effusion. Patient was prescribedifosfamide/Vp-16/methylene blue treatment. He received the same dosageamounts as in Examples 4-7, and over a total of six cycles neverdeveloped the symptoms associated with encephalopathy. After threecycles with ifosfamide, the patient had partial remission in the primaryand lymphnode metastases. After completion of six cycles, the patienthad partial remission of the lung, lymphnode and liver metastases.

Example 9

At the request of the inventors, an independent clinical analysis wasperformed at the Northern Centre for Cancer Treatment, Newcastle UponTyne, United Kingdom, on a patient using a combintation of ifosfamideand methylene blue. A female patient aged 35 years was diagnosed withadenocarcinoma of the oesophagus. She began chemotherapeutic treatmentand was administered ifosfamide, Mitomycin C, and cisplatin. Following asecond cycle of chemotherapy, the patient developed ifosfamideencephalopathy, becoming drowsy and confused. The patient eventuallyrecovered. Upon administration of a third cycle of treatment, thepatient was also given a prophylactic dosage of methylene blue.Following this, the patient remained conscious and alert throughouttreatment with no further signs of encephalopathy.

Example 10

100 ml of urine from a patient having had received methylene blue (300mg/day) and ifosfamide (3 mg/kg daily) was analysed. The urine wasrefluxed for 30 minutes with 2M hydrochloric acid in ethanol (50 ml).After sufficient heating, 50 mls each of H₂ O and 1M hexane were added,the mixture shaken. The organic phase was then reduced to dryness invacuo on a rotary evaporator. The dry residue was reconstituted in 1Mhexane (10 ml) and 1 μl aliquots were submitted to gaschromatography-mass spectrometry analysis (GCMS). A 16 meter OV-17capillary column was used with a temperature programme set for 40° C.for 1 minute, 5° C. per minute gradient to 250° C. and 250° C. for 10minutes. Electron impact mass spectra were obtained on the resultingchromatographic peaks of interest (See A, B, and C of FIG. 1). Thesepeaks were analysed through a Fisons Autospec Q MASS LIB GCMS computerlibrary of mass spectra developed by the Department of OrganicChemistry, University of Berne, Switzerland of known organic compounds(FIGS. 2-4). Identity of the three previously unknown eluting peaks wereunequivocally identified via computer matching as 2,2,2-trichlorethanal(FIG. 1 A), 3-chloroproprionic acid ethyl ester (FIG. 1 B), andtrichloracetic acid ethyl ester (FIG. 1 C).

Example 11

Human or bovine plasma (2.0 ml) containing 2-chlorethylamine (100 μg/ml)was incubated in a shaking water bath at 36° C. for 1 hour. Aliquots (5μl) were directly injected in to a Tenax packed gas chromatographiccolumn maintained at 120° C. in a Perkin Elmer 3920B gas chromatograph.The product of 2-chlorethylamine oxidation by plasma amine oxidase,2-chloracetaldehyde, was eluted at 9 minutes retention time. Externalcalibration curves were used to determine concentrations of resultingchloracetaldehyde. Similar experiments in which methylene blue (50 μM)was also added showed an inhibition of plasma amine oxidase by methyleneblue of 80%. The methylene blue is thus able to reduce the formation ofchloracetaldehyde from 2-chlorethylamine by the plasma to 20% of thatobserved in the absence of methylene blue.

While we have herein described a number of embodiments of thisinvention, it is apparent that the basic constructions can be altered toprovide other embodiments which utilise the methods of this invention.Therefore, it will be appreciated that the scope of this invention isdefined by the claims appended hereto rather than by the specificembodiments which have been presented herein by way of example.

What is claimed is:
 1. A method of treating or preventing toxicitycaused by ifosfamide, the method comprising administering to a subjectin need thereof an effective amount of a compound which is able tooxidize a reduced flavin moiety, wherein the compound is methylene blue.2. A method of treating or preventing ifosfamide encephalopathy, themethod comprising administering to a subject in need thereof aneffective amount of methylene blue.
 3. A method as claimed in claim 2,comprising administering at least 50 mg methylene blue.
 4. A method asclaimed in claim 3, comprising administering from 200 to 400 mgmethylene blue per day.
 5. A method as claimed in claim 4, comprisingadministering 300 mg methylene blue per day.
 6. A method as claimed inclaim 2, which further comprises administering fructose.
 7. A method asclaimed in claim 2, which further comprises administering glucose.